Trypsin detection film, preparation method therefor, application thereof, and trypsin detection kit

ABSTRACT

The present application provides a trypsin detection film, a preparation method therefor, application thereof and a trypsin detection kit, which relates to the technical field of trypsin detection. The preparation method for the trypsin detection film comprises the following steps: providing a polymer film substrate; immersing the polymer film substrate in a dye solution to attach a dye to the polymer film substrate, so as to obtain a trypsin detection film.

CROSS-REFERENCE OF RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No. 202010725938.5, filed Jul. 24, 2020, titled “Trypsin Detection Film, Preparation Method Therefor, Application Thereof, and Trypsin Detection Kit”, all of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

The present application relates to the field of trypsin detection, and more particularly to a trypsin detection film, a preparation method therefor, an application thereof and a trypsin detection kit.

BACKGROUND

Trypsin is a component of pancreatic juice and is an important protein digestive enzyme. Trypsin can limit the decomposition of precursors of chymotrypsinogen, procarboxypeptidase, phospholipase and other enzymes, and play a role in activation. Trypsin is a protease with the highest specificity, which can cleave the hydroxyl side of lysine and arginine residues in polypeptide chain, and is one of the most important digestive enzymes in protein decomposition. In clinical test, treatment and research, the content of trypsin is also an important marker for clinical diagnosis of pancreatic-related diseases. The secretion, activation, inhibition and imbalance of circulation of trypsin can lead to acute or chronic pancreatic diseases, such as pancreatic cancer. The detection of trypsin plays an important role in human health, production quality control and other fields. Therefore, the detection of trypsin is particularly important.

The traditional method for detecting the content of trypsin is mainly spectrophotometry, but this method has many steps, is complicated in operation and has low detection sensitivity. At present, the main methods for determination of trypsin include UV spectrophotometry, colorimetry, fluorometry, immunoassay, radio-element labeling, mass spectrometry (MS), high performance liquid chromatography (HPLC), electrophoresis, etc. Among them, UV spectrophotometry and colorimetry are simple, but have low sensitivity and poor reproducibility. Immunoassay is highly sensitive, but because of the use of monoclonal antibodies, the cost is also high, and certain operating techniques are required, so it is difficult to popularize. Radio-element labeling method needs to label the substrate, which is complicated to prepare and has certain safety problems. The mass spectrometer in mass spectrometry is expensive and requires high purity of the sample. Chromatography and electrophoresis also require relatively complicated operations and long detection times.

SUMMARY OF THE INVENTION

An object of the present application is to provide a trypsin detection film, a preparation method therefor, an application thereof and a trypsin detection kit, which have the advantages of easy operation, short time, low cost, low technical threshold and the like, and can overcome the above problems or at least partially solve the above technical problems.

In order to achieve the object, the technical solution adopted by the present application is as follows.

According to one aspect of the present application, a preparation method for a trypsin detection film is provided, comprising the following steps:

providing a polymer film substrate;

immersing the polymer film substrate in a dye solution to attach a dye to the polymer film substrate, so as to obtain a trypsin detection film.

Where the dye interacts with trypsin, and the color of the dye is different in an environment with different trypsin concentration; the dye comprises at least one of a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a triarylmethane dye, a xylenol orange dye or a metal complex dye.

In one possible embodiment, the concentration of the dye solution is between 0.25 mg/mL and 2.5 mg/mL, preferably between 1 mg/mL and 2 mg/mL;

preferably, a solvent of the dye solution comprises water and/or an alcoholic solvent.

Preferably, the polymer film substrate is immersed in the dye solution at a temperature of 20-40° C., preferably 25-35° C.; and/or the polymer film substrate is immersed in the dye solution for a period of 15-30 min, preferably 18-22 min.

In one possible embodiment, the preparation method for the polymer film substrate comprises the following steps:

providing a base plate assembly comprising a base plate and a first lubricant disposed on the base plate;

placing a film-forming solution of the polymer film substrate on the base plate assembly, and putting a cover plate with a second lubricant on the film-forming solution, where the film-forming solution is in contact with the first lubricant and the second lubricant, respectively; and

separating the polymer film substrate from the base plate assembly and the cover plate after a polymerization reaction of various raw materials in the film-forming solution to obtain the polymer film substrate.

Preferably, the base plate assembly further comprises a tinfoil, where the tinfoil is arranged on the base plate, and the first lubricant is arranged on the tinfoil.

In one possible embodiment, the first lubricant and the second lubricant are each independently selected from at least one of white vaseline, silicone oil, paraffin, mineral oil or grease.

In one possible embodiment, the polymerization reaction is carried out under irradiation of ultraviolet light, where the wavelength of the ultraviolet light is preferably 250-400 nm, and/or the time of ultraviolet light irradiation is preferably 10-30 min.

Preferably, after ultraviolet irradiation, the polymer film substrate is placed in a curing chamber for 5-10 min.

In one possible embodiment, separating the polymer film substrate from the base plate assembly and the cover plate comprises: removing the base plate assembly, placing the cover plate attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the cover plate to obtain the polymer film substrate; or, removing the cover plate, placing the base plate assembly attached with the polymer film substrate in the standing solution, standing for 10-30 min, and removing the base plate assembly to obtain the polymer film substrate;

preferably, removing the base plate, placing the cover plate and a tinfoil attached with the polymer film substrate in the standing solution, standing for 10-30 min, and removing the tinfoil to obtain the polymer film substrate.

Preferably, after separating the polymer film substrate from the base plate assembly and the cover plate, the method further comprises the step of cleaning the polymer film substrate, comprising ultrasonic cleaning in clear water, an alcohol solution and clear water in sequence.

In one possible embodiment, the preparation method for the film-forming solution comprises:

uniformly mixing an ionic liquid monomer and a base film monomer, adding a cross-linking agent and an initiator, and then carrying out a second ultrasonic treatment to obtain the film-forming solution.

Preferably, the irradiation time of the second ultrasonic treatment is 10-30 min.

Preferably, the preparation method for the film-forming solution further comprises performing a first ultrasonic treatment on the ionic liquid monomer, where the first ultrasonic treatment lasts for 10-30 min.

Preferably, the film-forming solution comprises at least one of an imidazole ionic liquid, a pyridine ionic liquid, a quaternary ammonium salt ionic liquid, a quaternary phosphine ionic liquid or a pyrrolidine ionic liquid.

Preferably, the ionic liquid monomer comprises bromobutane and vinylimidazole. Preferably, a molar ratio of bromobutane to vinylimidazole is 2:1 to 1:1.

Preferably, the base film monomer comprises acrylonitrile. Preferably, the mass of the acrylonitrile is greater than or equal to the sum of the mass of the bromobutane and the vinylimidazole.

Preferably, the cross-linking agent comprises N,

N′-methanediylbisprop-2-enamide. Preferably, the mass of the cross-linking agent is 8 wt %-12 wt % of the total mass of the bromobutane, the vinylimidazole and the acrylonitrile.

Preferably, the initiator comprises

Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide. Preferably, the mass of the initiator is 1 wt %-4 wt % of the total mass of the bromobutane, the vinylimidazole and the acrylonitrile.

According to another aspect of the present application, a trypsin detection film is provided, which is prepared by the preparation method for the trypsin detection film.

According to another aspect of the present application, an application of a detection film prepared by the preparation method for the trypsin detection film or of the trypsin detection film in detecting trypsin is provided, and the application comprises:

placing the trypsin detection film in a liquid to be detected, so that the dye in the trypsin detection film contacts or interacts with the liquid to be detected.

In one possible embodiment, the application further comprises: observing a color change of the trypsin detection film.

In one possible embodiment, the application further comprises: determining the concentration of the trypsin according to different colors shown by the trypsin detection film, where the trypsin detection film has different colors in an environment of the liquid to be detected containing different concentration of trypsin.

According to another aspect of the present application, a trypsin detection kit is provided, which comprises a detection film prepared by the preparation method for the trypsin detection film or the trypsin detection film.

In one possible embodiment, the trypsin detection kit further comprises a standard colorimetric card for trypsin detection.

Compared with the prior art, the technical solution provided by the present application can achieve the following beneficial effects.

The preparation method for the trypsin detection film has the characteristics of simplicity and convenience in synthesis, short preparation period, easy operation, low cost, low technical threshold and the like, and is easy to realize large-scale production and use. In addition, the trypsin detection film prepared by the method can be applied to trypsin detection on a large scale, so that the problems of large technical difficulty, long detection time, high cost and incapability of meeting current requirements of the existing trypsin detection mode are solved.

The trypsin detection kit of the present application has all the features and advantages of the trypsin detection film described above, and will not be described here.

It should be understood that the above description and the details to be set forth in the following text are only exemplary, which are not intended to limit the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art.

FIG. 1 is a contrast relationship table of RGB color values of a trypsin detection film according to an embodiment of the present application.

DETAILED DESCRIPTION

In order to make the object, technical solution and advantages of the present application clearer, the technical solution of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art without creative work based on the technical solutions and embodiments provided in the present application belong to the protection scope of the present application. The embodiments in which specific conditions are not indicated are carried out under conventional conditions or conditions suggested by the manufacturer.

It should be noted that the term “and/or” or the “/” used herein only means an association relationship describing associated objects, indicating that there can be three types of relationships. For example, A and/or B can refer to: only A exists, both A and B exist, and only B exists. The singular forms “a”, “said” and “the” used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

The endpoints of ranges and any values disclosed herein are not limited to the precise ranges or values, and such ranges or values should be understood to include values approaching such ranges or values. For numerical ranges, between endpoints of individual ranges, between endpoints or individual point values of individual ranges, and between individual point values may be combined with each other to yield one or more new numerical ranges.

All the technical features and preferred features mentioned herein can be combined with each other to form new technical solutions unless otherwise specified. Unless otherwise defined or indicated, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.

As mentioned in the background, the existing trypsin detection methods have certain defects more or less. For example, the prior art discloses a biosensor for detecting trypsin, a preparation method therefor and an application thereof in detecting trypsin, where the biosensor comprises a magnetic gold electrode, magnetic nanoparticles, a polypeptide chain 1 and a polypeptide chain 2. The surface of the magnetic nanoparticles is modified with carboxyl, the surface of the polypeptide chain 2 is loaded with silver nanoparticles, one terminal sequence of the polypeptide chain 1 and one terminal sequence of the polypeptide chain 2 are both VIA, and the polypeptide chain 1 contains an arginine residue. The surface of the detection terminal of the magnetic gold electrode magnetically adsorbs the magnetic nanoparticles, the polypeptide chain 1 is fixed on the magnetic nanoparticles through the reaction of amino at the terminal and carboxyl modified on the surface of the magnetic nanoparticles, and the polypeptide chain 2 is self-assembled on the polypeptide chain 1 through the terminal sequence VIA. Although the sensor and the method can realize the detection of trypsin, the process is cumbersome, difficult to prepare, and susceptible to interference. For another example, the prior art discloses a fluorescent probe for trypsin detection and a preparation method thereof, where the fluorescent probe is a composite aqueous solution composed of a fluorescently labeled polymer and cytochrome C adsorbed on the surface of the fluorescently labeled polymer, the weight percentage concentration of the composite aqueous solution is 0.0005%-0.05%. The water-soluble fluorescently labeled polymer obtained by modifying hydrophobic dye pyrene with a hydrophilic polymer is used as an energy donor for fluorescence emission. Cytochrome C is used as a substrate of trypsin, a fluorescent probe for detecting trypsin is obtained through adsorption of the cytochrome C on the surface of the fluorescently labeled polymer, and the detection of enzyme activity is realized through the change of fluorescence intensity. Although the probe can achieve the detection of trypsin, the process is cumbersome, the adsorption rate is low, the preparation is difficult, and the resistance to interference is weak, the fluorescence is easily quenched, while the detection of fluorescence intensity requires specialized equipment. For another example, the prior art discloses a colorimetric analysis method for rapid determination of trypsin, which comprises the following steps: firstly, detecting the UV-Vis (ultraviolet-visible spectrum) of silver nanoparticles, then carrying out a chromogenic reaction between trypsin and polypeptide-modified silver nanoparticles, detecting the ultraviolet spectrum of a reaction product after the chromogenic reaction is completed, and then calculating the difference between peak absorbance values of the silver nanoparticles and the reaction product, and comparing the difference with a predetermined standard curve to obtain the content of trypsin. Although the detection method has high sensitivity, low detection limit and no need of complex chemical modification or signal labeling, it requires a high level of detection personnel, specialized equipment, and silver nanoparticles are easily oxidized and not easily preserved.

Therefore, the existing trypsin detection method has the defects of high technical difficulty, complex test, high cost, single function, and incapability of meeting current requirements, etc. In some embodiments, the present application provides a trypsin detection film, a preparation method therefor, an application thereof and a trypsin detection kit. According to the present application, the trypsin detection film is prepared by a simple and efficient preparation method, and has the advantages of simplicity and convenience in synthesis, short preparation period, low technical threshold, capability of being reused and the like.

In a first aspect, in some embodiments, there is provided a preparation method for a trypsin detection film, comprising the following steps:

providing a polymer film substrate;

immersing the polymer film substrate in a dye solution to attach a dye to the polymer film substrate, so as to obtain the trypsin detection film;

where, the dye is suitable for interacting with trypsin, and the color of the dye is different in the environment with different trypsin concentration; the dye comprises at least one of a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a triarylmethane dye, a xylenol orange dye or a metal complex dye.

The method has the advantages of simple preparation, easy operation, low cost and easy scale production and use.

Specifically, the preparation method for the trypsin detection film is a simple, convenient and efficient preparation method, and has the advantages of simple preparation, easy operation, low technical threshold, low cost, low requirements on detection and operator, easy large-scale production and use. The trypsin detection film prepared by the method can be applied to trypsin detection, which has the advantages of simple and convenient operation, low cost and safety, etc., and can determine the concentration of trypsin based on color, and thus has great potential for application.

In the trypsin detection film, the polymer film substrate can be a polyionic film, namely a polyionic liquid type film, which has the excellent performances of an ionic liquid and a polymer, can overcome the fluidity of the ionic liquid, has unique physical and chemical properties, and can be well applied to the field of medical detection.

In the trypsin detection film, the dye is a compound which has a certain color and can enable other substances to obtain bright or obvious color. In the embodiments of the present application, the dye can change color when trypsin is detected, and the dye and the polymer film substrate can form good stable coordination. Specifically, the polymer film substrate can adsorb the dye, or the dye can be well attached to the polymer film substrate, and the dye can be well maintained on the polymer film substrate due to strong ionic interaction between the polymer film substrate and the dye.

Compared with the prior art, in the embodiments of the present application, the dye and the trypsin combine or react (interact) for color development, and the method has the advantages of simple operation, low technical threshold, obvious color change, low cost, and fast detection.

Specifically, the specific shape of the trypsin detection film may be of various types, for example, it may be any shape such as a circle, a square, a polygon or other irregular shapes, or it may be a sheet, a strip, etc. The embodiments of the present application do not limit the specific shape of the trypsin detection film.

Specifically, the size of the trypsin detection film can also be adjusted according to actual conditions, and the embodiments of the present application do not limit the specific size of the trypsin detection film.

Specifically, the dye is suitable for interaction with trypsin, and the color of the dye varies in environments with different concentrations of trypsin. That is, the dye has different color when the concentration of trypsin is different. Therefore, the interaction between the dye and the trypsin is utilized for color development, and the detection result can be obtained by observing the color change of the trypsin detection film. In addition, different color development effect can be achieved by mixing the dyes or changing the concentration of the dyes.

Specifically, the dye includes, but is not limited to, at least one of a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a triarylmethane dye, a xylenol orange dye, or a metal complex dye.

It should be noted that the dye can be selected from the bromocresol purple dye, or the 3, 3′, 5, 5′-tetramethylbenzidine dye, or the xylenol orange dye, or the triarylmethane dye, or the metal complex dye, or mixture of the above dyes, but is not limited thereto. More generally, the dye may be any dye satisfying the following three conditions: (1) can be stably combined with a polymer film substrate; (2) capable of binding or interacting with trypsin; (3) meet the requirements of biological safety.

The bromocresol purple dye, taken as an example, belongs to a biological dye. The dye is safe and non-toxic, and can be combined with trypsin through a non-covalent bond. A hydrophobic core of the trypsin is combined with a non-polar group of the bromocresol purple dye. In addition, the volume of an aggregate after combination is larger than the volume of the bromocresol purple dye, so that the molar absorptivity after combination is changed, the light scattering signal is changed, and different colors are displayed. In addition, the intensity of the light scattering signal is in direct proportion to the number of particles in unit volume, namely, is in direct proportion to the concentration of trypsin, so the detection of the concentration of trypsin can be realized by observing colors. In the detection process, the bromocresol purple dye in the trypsin detection film shows different colors for trypsin with different concentrations, and the trypsin concentration can be quantitatively detected through color change.

Specifically, in the trypsin detection film, in one aspect, the polymer film substrate can be a carrier or platform of a dye. Due to the strong ionic interaction between the polymer film substrate and the dye, especially the bromocresol purple dye, the 3, 3′, 5, 5′-tetramethylbenzidine dye, and the xylenol orange dye, etc., the dye can be well maintained or attached to the polymer film substrate, namely the dye can be stably combined with the polymer film substrate. In another aspect, the dye, especially the bromocresol purple dye, the 3, 3′, 5, 5′-tetramethylbenzidine dye, and the xylenol orange dye, etc., is safe and non-toxic, and can be combined with trypsin to change the color of the trypsin detection film, for example, the color can be changed from blue to yellow, and the depth (tone) of the color change is different according to different concentrations of the trypsin, so that the condition of the trypsin can be quantitatively detected through the color change. Alternatively, in other embodiments of the present application, the dye may also change color due to other types of physical or chemical reactions between trypsin and the dye, which are not described herein.

As can be seen from the above, the embodiments of the present application directly utilize a dye, such as the bromocresol purple dye, the 3, 3′, 5, 5′-tetramethylbenzidine dye or the xylenol orange dye, etc., to combine or react with trypsin to achieve the purpose of changing the color of the dye, thus achieving the detection effect. In addition, the concentration of trypsin can be determined by the change degree before and after detection. For example, the concentration of trypsin in liquid environment can be determined by the change of color shade (tone), so as to assist in examination and corresponding treatment. The trypsin detection film is applied to detection of trypsin, and has the characteristics of simple operation, obvious color change, fast detection, high efficiency, low requirements on detection personnel, etc.

In some embodiments, the polymer film substrate comprises a polyionic liquid film (polyionic film) containing ionic liquid.

Preferably, the ionic liquid comprises, but is not limited to, at least one of an imidazole ionic liquid, a pyridine ionic liquid, a quaternary ammonium salt ionic liquid, a quaternary phosphine ionic liquid or a pyrrolidine ionic liquid. It can be understood that, according to the function of the ionic liquid, the ionic liquid used for preparing the polymer film substrate can be a functionalized ionic liquid commonly used in the art. Exemplarily, the ionic liquid may be the imidazole ionic liquid, the pyridine ionic liquid, the quaternary ammonium salt ionic liquid, or the pyrrolidine ionic liquid, etc. The embodiments of the present application are not limited to the specific type of the ionic liquid, and the ionic liquids listed above can be used, and other types of ionic liquids known in the art can also be used.

Preferably, an ionic liquid monomer forming the polyionic liquid film comprises bromobutane and vinylimidazole. It can be understood that the bromobutane and the vinylimidazole can react and form the ionic liquid monomer.

Preferably, a base film monomer for forming the polyionic liquid film comprises, but is not limited to, acrylonitrile. The base film monomer may be acrylonitrile, or a mixture of acrylonitrile and styrene, or other base film monomers commonly used in the art with similar functions or effects.

Preferably, the cross-linking agent for forming the polyionic liquid film includes, but is not limited to, N, N′-methylenebisprop-2-enamide (MBA for short). The MBA used as a cross-linking agent plays a bridging role among molecular monomers. Molecules are mutually bonded and cross-linked into a net structure, promoting the bonding among polymer molecular chains. In addition, the cross-linking agent can also be other cross-linking agents with similar properties or functions commonly used in the art.

Preferably, an initiator for forming the polyionic liquid film includes, but is not limited to, Diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide (TPO for short). TPO is a photoinitiator, which is a light yellow solid and is mainly used for photocuring. The initiator is a high-efficiency universal ultraviolet photoinitiator and can be used to initiate UV polymerization reactions of unsaturated prepolymer system. In addition, the initiator can also be other initiators with similar properties or functions commonly used in the art. For example, the initiator may be an initiator commonly used in the art, such as photoinitiator 907, photoinitiator 184, azobisisobutyronitrile, benzoin and derivatives thereof. Those skilled in the art can select an appropriate initiator depending on the specific type of ionic liquid monomer, base film monomer, etc.

It should be noted that the embodiments of the present application are not limited to the specific type of the ionic liquid, the specific type of the base film monomer, the specific type of the cross-linking agent and the specific type of the initiator, and various types commonly used in the art can be used as long as the object and application scenarios of the present application are not limited. For the convenience of description, the embodiments of the present application mainly take MBA as a cross-linking agent, TPO as an initiator, acrylonitrile as a base film monomer, and an ionic liquid monomer formed by bromobutane and vinylimidazole as an example to specifically describe the trypsin detection film and the preparation method thereof. However, those skilled in that art will appreciate that the principles disclosed herein may be implemented in any suitable polymer film substrate material. Further, descriptions of well-known functions or actions may be omitted for clarity and conciseness.

Preferably, the concentration of the dye solution is 0.25-2.5 mg/mL, preferably 1-2 mg/mL; typically but not limiting, for example, may be 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 2.5 mg/mL, etc. The dye solution with proper concentration range can make the final display effect better, which is more beneficial to accurately detect the concentration of trypsin.

Preferably, a solvent of the dye solution comprises water and/or an alcoholic solvent. For example, the solvent may be a lower alcohol, and may further be an alcohol solvent such as ethyl alcohol. That is, the dye solution may be formed by dissolving a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a xylenol orange dye, or the like in water or in an alcoholic solvent.

Preferably, the temperature at which the polymer film substrate is immersed in the dye solution is in the range of 20-40° C., preferably 25-35° C. The immersion temperature may be room temperature, for example, 20° C., 25° C., 30° C., 40° C., or the like.

Preferably, the polymer film substrate is immersed in the dye solution for a period of 15-30 min, preferably 18-22 min. Typically but not limiting, for example, may be 15 min, 20 min, 22 min, 25 min, 30 min, etc. Therefore, the polymer film substrate can be better combined with the dye in a proper time and temperature range, the property of the dye is not changed, and the obtained trypsin detection film has good performance and high efficiency.

In some embodiments, the preparation method for the polymer film substrate comprises the following steps:

providing a base plate assembly comprising a base plate and a first lubricant disposed on the base plate;

placing a film-forming solution of the polymer film substrate on the base plate assembly, and putting a cover plate with a second lubricant on the film-forming solution, where the film-forming solution is in contact with the first lubricant and the second lubricant, respectively; and

separating the polymer film substrate from the base plate assembly and the cover plate after a polymerization reaction of various raw materials in the film-forming solution to obtain the polymer film substrate.

It will be appreciated that after the polymerization reaction has taken place and prior to separation, the obtained is a composite structure comprising the polymer film substrate, the cover plate and the base plate assembly, and then, via a separation step, the polymer film substrate is separated from the cover plate and the base plate assembly to obtain the polymer film substrate.

In some embodiments, the base plate assembly further comprises a tinfoil, where the tinfoil is arranged on the base plate, and the first lubricant is arranged on the tinfoil. It should be understood that that base plate assembly may comprise the base plate and the first lubricant, or may comprise the base plate, the tinfoil and the first lubricant. When the base plate assembly comprises a tinfoil, the tinfoil needs to be flatly laid on the substrate, or the tinfoil flatly wraps the base plate, and then the first lubricant can be disposed on the tinfoil, which is more beneficial to the preparation of the polymer film substrate.

According to the embodiments of the present application, the preparation method for the polymer film substrate has the characteristics of high yield, high reaction rate and high energy efficiency. Meanwhile, the whole process is simple and convenient to operate, low in requirement on experimental equipment and high in processing safety. In addition, the polymer film substrate prepared by the preparation method has stable structure, is easy to peel and has low irritation.

According to the preparation method for the polymer film substrate, through the use of the tinfoil and the lubricant, a barrier is formed between the film-forming solution and the cover plate and/or the base plate, so that the film-forming solution before film forming cannot be directly contacted with the base plate and/or the cover plate, reducing the influence of the cover plate and/or the base plate on the film forming quality in the film forming process, thus ensuring the integrity and the thickness uniformity of the polymer film substrate, and greatly shortening the time required for separating the polymer film substrate.

Specifically, the base plate may be a glass plate, a stainless steel plate, a hard plastic plate through which ultraviolet light does not easily pass, etc. Among these base plates, it is preferable that the base plate is a glass plate. The glass plate has the advantages of easily available raw materials and low cost, and is good in heat resistance, and easy to cool after polymerization reaction is carried out by ultraviolet irradiation, so that the operation time is shortened.

Specifically, the cover plate may be a glass plate or a hard plastic plate, etc. Preferably, the cover plate may be a transparent glass plate or a transparent hard plastic plate. The transparent glass plate or the transparent hard plastic plate is used for enabling the polymerization reaction to be carried out under the irradiation of ultraviolet light, so that the reaction speed is accelerated, and an optional method is provided for carrying out the polymerization reaction.

Specifically, the lubricant used, such as the first lubricant or the second lubricant, needs to be inert to ultraviolet light and not interfere with ultraviolet light. The first lubricant and the second lubricant may be of the same type or different types.

In some embodiments, the first lubricant and the second lubricant are each independently selected from at least one of white petrolatum, silicone oil, paraffin, mineral oil, or grease. Exemplarily, the first lubricant may be white vaseline, may be silicone oil, may be mineral oil, etc., and the second lubricant may be white vaseline, may be silicone oil, may be grease, etc.

In the field of medical devices, the lubricant used needs to be non-toxic, corrosion-free, residue-free and transparent. Specifically, the fields with high requirements on safety, such as sensors and drug controlled release, have higher requirements on lubricants, and common medical-grade lubricants or food-grade lubricants can be adopted.

Specifically, in the preparation method for the polymer film substrate, a film-forming solution may be prepared in advance. Then, the tinfoil is flatly laid on the base plate, or the tinfoil flatly wraps the base plate, or the tinfoil can be wiped with a lint-free cloth until the tinfoil has no wrinkles. The smooth surface of the tinfoil is put upward, then a first lubricant such as white vaseline is smeared on the tinfoil, and the lint-free cloth is used to wipe until the surface is smooth. Then, the prepared film-forming solution is covered on the tinfoil with the first lubricant, and is uniformly coated. Then, a cover plate coated with the second lubricant in advance is covered on the tinfoil carrying the film-forming solution. It can be understood that the surface of the film-forming solution may be in contact with the second lubricant and the first lubricant.

Alternatively, the base plate is wetted with a wetting solution before the tinfoil is laid on the base plate. The purpose of wetting the base plate is to expel air between the base plate and the tinfoil. In addition, the bonding force between the base plate and the tinfoil is increased through the adhesion of the wetting solution and the tinfoil, so that it is easier to flatten the tinfoil, and thereby improve the flatness of the surface of the tinfoil. There may be various wetting solutions for wetting the base plate, but the wetting solution is preferably water, ethyl alcohol, or a mixed solution thereof from the viewpoint of source, cost, and environmental protection.

In addition, in other embodiments, the base plate assembly may comprise only a base plate. Specifically, in the preparation method for the polymer film substrate, a film-forming solution can be prepared in advance, then a first lubricant such as white vaseline is coated on the base plate, and the first lubricant is wiped by a lint-free cloth until the surface is smooth. After that, the prepared film-forming solution is covered on the base plate with the first lubricant, evenly coated, and then the cover plate coated with the second lubricant in advance is covered on the base plate carrying the film-forming solution. It can be understood that the surface of the film-forming solution may be in contact with the second lubricant and the first lubricant.

Specifically, in some embodiments, the preparation method for the film forming comprises:

uniformly mixing an ionic liquid monomer and a base film monomer, adding a cross-linking agent and an initiator, and then carrying out a second ultrasonic treatment to obtain the film-forming solution;

preferably, the time of the second ultrasonic treatment is 10-30 min; typically but not limiting, for example, may be 10 min, 15 min, 20 min, 25 min, 30 min, etc.

Preferably, the preparation method for the film-forming solution further comprises performing a first ultrasonic treatment on the ionic liquid monomer, wherein the first ultrasonic treatment lasts for 10-30 min; typically but not limiting, for example, may be 10 min, 15 min, 20 min, 25 min, 30 min, etc.

In the preparation of the film-forming solution, compared with the existing “two-step method” for preparing the ionic liquid, the preparation method provided by the embodiment of the present application can obviously improve the preparation efficiency of the film-forming solution by using an ultrasonic method, greatly shorten the preparation time, and is easy to operate and good in controllability. Therefore, the preparation efficiency of the polymer film substrate and the trypsin detection film can be further improved by adopting the preparation method for the film-forming solution.

According to the embodiments of the present application, different film-forming solutions can be selected according to different requirements. The component of the ionic liquid in the film-forming solution may be one or more of the imidazole ionic liquid, the pyridine ionic liquid, the quaternary ammonium salt ionic liquid, the quaternary phosphine ionic liquid or the pyrrolidine ionic liquid, or may also be other types of ionic liquids well known in the art.

Preferably, the ionic liquid monomer comprises bromobutane and vinylimidazole.

Preferably, the base film monomer comprises acrylonitrile.

Preferably, the cross-linking agent comprises N, N′-methanediylbisprop-2-enamide (MBA).

Preferably, the initiator comprises Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO).

Specifically, in the preparation of the film-forming solution, the molar ratio of bromobutane to vinylimidazole may be 2:1 to 1:1, which may be, for example, 1:1. To ensure complete reaction, the mass of the acrylonitrile needs to be greater than or equal to the sum of the mass of the bromobutane and vinylimidazole. In consideration of the reaction ratio, the conversion rate, the dosage and the subsequent cleaning treatment process of the three components, the mass of the acrylonitrile is preferably the sum of the mass of the bromobutane and the vinylimidazole. The mass of the cross-linking agent is 8 wt %-12 wt % based on the total mass of bromobutane, vinylimidazole, and acrylonitrile, and may be, for example, 8 wt %, 9 wt %, 10 wt %, 12 wt %, etc. The mass of the initiator is 1 wt %-4 wt % of the total mass of the bromobutane, the vinylimidazole and the acrylonitrile; for example, it may be 1 wt %, 1.5 wt %, 2 wt %, 3 wt %, 4 wt %, etc. It should be understood that the ratio of the above raw materials is not limited thereto, and can be appropriately adjusted according to the actual process conditions.

Specifically, the preparation of the film-forming solution may include the following steps:

mixing the bromobutane and the vinylimidazole in equal molar ratio, and carrying out an ultrasonic treatment on the obtained mixed solution for 15 min until the two are fully mixed;

adding an amount of the acrylonitrile equal to the total mass of the bromobutane and the vinylimidazole after removing impurities;

then, adding MBA with a mass ratio of 8 wt % calculated by the total mass of the bromobutane, the vinylimidazole and the acrylonitrile and TPO with a mass ratio of 1 wt % calculated by the total mass of the bromobutane, the vinylimidazole and the acrylonitrile, and after the addition, carrying out ultrasonic treatment for 15 min to obtain a film-forming solution, where the obtained film-forming solution is a clear transparent solution.

In this preparation step, ultrasonic treatment can increase the energy field for the mixed solution of the bromobutane and the vinylimidazole, thus accelerating the reaction.

Further, in the preparation method for the trypsin detection film, raw materials in the film-forming solution are subjected to polymerization reaction, and in some embodiments, the polymerization reaction is carried out under the irradiation of ultraviolet light.

The wavelength of the ultraviolet light is preferably 250-400 nm; typically but not limiting, for example, may be 250 nm, 300 nm, 325 nm, 400 nm, etc.

The irradiation time of ultraviolet light is preferably 10-30 min; typically but not limiting, for example, may be 10 min, 20 min, 30 min, etc.

Alternatively, the polymerization reaction may be carried out under heating while being carried out under irradiation of ultraviolet light. Where, the heating temperature may be 20-60° C., and typically, but not limiting, for example, may be 20° C., 30° C., 40° C., 50° C., 60° C., etc.

Polymerization reaction under this condition is conducive to obtaining the polymer film substrate with excellent performance and high efficiency.

The polymerization reaction may be initiated by an initiator. The initiator may be a common initiator in the art, such as photoinitiator 907, photoinitiator 184, azobisisobutyronitrile, benzoin and derivatives thereof, etc. Those skilled in the art may select an appropriate initiator according to the specific components of the film-forming solution.

In addition, in the preparation method for the trypsin detection film, after the polymerization reaction of various raw materials in the film-forming solution, a separation is performed (for example, the polymer film substrate is separated from the cover plate and/or the base plate assembly). In some embodiments, the method of separation includes: removing the base plate assembly, placing the cover plate attached with the polymer film substrate in a standing solution and standing for 10-30 min, to make the polymer film substrate automatically separated from the cover plate in the standing solution to obtain the polymer film substrate;

or, removing the cover plate, placing the base plate assembly attached with the polymer film substrate in the standing solution, and standing for 10-30 min to make the polymer film substrate separated from the base plate assembly in the standing solution, removing the base plate assembly, and obtaining the polymer film substrate.

Further, when separating the polymer film substrate from the base plate assembly, since the tinfoil and the first lubricant are arranged between the polymer film substrate and the base plate as a barrier, the base plate and the tinfoil attached with the polymer film substrate can be easily separated. Compared with the method for separating the base plate from the polymer film substrate, the tinfoil is very thin, and the first lubricant is arranged between the tinfoil and the polymer film substrate, so that the tinfoil and the polymer film substrate are easier to separate. Specifically, in the process of separating the polymer film substrate and the tinfoil, the tinfoil and the polymer film substrate can be placed in a solution to make the two naturally separated by the solution, or the tinfoil and the polymer film substrate can be separated by a mechanical method (such as manual peeling of the tinfoil), so as to obtain a complete polymer film substrate.

Alternatively, the standing solution may be water or other solvents.

In such a way, the polymer film substrate can be automatically separated from the cover plate or the base plate or the tinfoil, which is simple in operation, while improving the yield rate and production efficiency.

Alternatively, the polymer film substrate obtained by separation is cleaned, for example, by ultrasonic cleaning in clean water, absolute ethyl alcohol and clean water in sequence.

It can be seen from the above that, according to the preparation method in the embodiments of the present application, by using the tinfoil, the first lubricant and the second lubricant, not only the integrity of the polymer film substrate and the uniformity and stability of the film thickness can be ensured, but also the polymer film substrate can be separated more easily, that is, the automatic peeling of the polymer film substrate is realized, and the time required for separation is greatly shortened. Meanwhile, preparing the film-forming solution by ultrasonic method also greatly shortens the time of preparation, and the operation is simple and efficient. Therefore, compared with the existing film preparation method, this preparation method for the polymer film substrate has the characteristics of easy operation, high efficiency, short time, etc.

Therefore, the preparation method for the trypsin detection film provided by present application in some embodiments has the advantages of simplicity and convenience in synthesis, easiness in operation, low technical threshold, easiness in separation of the polymer film substrate, short preparation time, high preparation efficiency, easiness in realizing large-scale production and the like, which is a simple, convenient and efficient preparation method, and is easy for large-scale production and application.

In a second aspect, in some embodiments, there is provided a trypsin detection film, which is prepared by the preparation method for the trypsin detection film.

The trypsin detection film comprises:

a polymer film substrate; and a dye attached to the polymer film substrate.

In some embodiments, the dye is suitable for interaction with trypsin, and the color of the dye varies in environments with different concentrations of trypsin.

In some embodiments, the dye includes, but is not limited to, at least one of a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a triarylmethane dye, a xylenol orange dye, or a metal complex dye.

In some embodiments, the polymer film substrate comprises a polyionic liquid film comprising an ionic liquid.

Preferably, the ionic liquid comprises, but is not limited to, at least one of an imidazole ionic liquid, a pyridine ionic liquid, a quaternary ammonium salt ionic liquid, a quaternary phosphine ionic liquid or a pyrrolidine ionic liquid.

Preferably, the ionic liquid monomer forming the polyionic liquid film comprises, but are not limited to, bromobutane and vinylimidazole.

Preferably, the base film monomer for forming the polyionic liquid film includes, but is not limited to, acrylonitrile.

Preferably, the cross-linking agent for forming the polyionic liquid film includes, but is not limited to, MBA.

Preferably, the initiator for forming the polyionic liquid film includes, but is not limited to, TPO.

It should be understood that for the same or similar parts in the preparation method for the trypsin detection film according to the second aspect and the preparation method for the trypsin detection film according to the first aspect, the description of the preparation method for the trypsin detection film of the first aspect can be a reference, and will not be described here.

In a third aspect, in some embodiments, an application of a detection film prepared by the preparation method for the trypsin detection film or of the trypsin detection film in detection of trypsin is provided. The application comprises the following steps:

placing the trypsin detection film in a liquid to be detected, so that the dye in the trypsin detection film contacts or interacts with the liquid to be detected.

In some embodiments, the application further comprises: observing a color change of the trypsin detection film.

In some embodiments, the application further comprises: determining the concentration of the trypsin according to different colors shown by the trypsin detection film, where the trypsin detection film has different colors in an environment of the liquid to be detected containing different concentration of trypsin.

It should be noted that the display of the color of the trypsin detection film may also be related to the immersion time or immersion temperature. Specifically, for example, in the case where the polymer film substrate is immersed in the dye solution for the same time, and in the case where the polymer film substrate is immersed in the dye solution at the same temperature, the color change of the trypsin detection film before and after the detection is affected by the trypsin concentration. When the polymer film substrate is immersed in the dye solution for different time or at different temperature, the color change of the trypsin detection film before and after detection may be affected by the trypsin concentration and the immersion time or the immersion temperature. Therefore, the time when and the temperature at which the polymer film substrate is immersed in the dye solution need to be within an appropriate range, or the time when and the temperature at which the polymer film substrate is immersed in the dye solution need to be the same when detecting a series of trypsin concentrations, so as to minimize the influence of immersion time and immersion temperature on the color change of the trypsin detection film.

The application of the trypsin detection film as described in the embodiments of the present application in detection of trypsin has the advantages of easiness in operation, obvious change before and after detection, high detection speed and high efficiency, etc., and can alleviate the problems of complex equipment, high technical requirements, complex detection, high cost and the like of the existing trypsin detection method.

It should be understood that, for the same or similar parts in the application of the trypsin detection film according to the third aspect as the trypsin detection film and the preparation method according to the first and second aspects, reference may be made to the description of the trypsin detection film and the preparation method, which will not be repeated here.

In a fourth aspect, in some embodiments, a trypsin detection kit is provided comprising the aforementioned trypsin detection film.

As can be seen from the above, the trypsin detection kit according to the embodiments of the present application comprises the trypsin detection film, and thus has at least the same advantages as the trypsin detection film and the preparation method and the application thereof, which are not described herein. The trypsin detection kit works mainly by combination or reaction between the dye in the trypsin detection film and the trypsin to show color. When the concentration of trypsin varies, the trypsin detection film has different colors, so the concentration of trypsin can be determined by the change in color of the trypsin detection film before and after the detection.

In some embodiments, the trypsin detection kit further comprises a standard colorimetric card for trypsin detection, or a standard color scale for trypsin detection.

Further, the trypsin detection kit can further comprise a packaging box and a user guide.

The trypsin detection kit can be provided with a packaging box, and a trypsin detection film and a standard colorimetric card can be placed in the packaging box, and a user guide can also be placed in the packaging box. Where, the number of the trypsin detection films may not be limited, for example, 1-5 trypsin detection films or more trypsin detection films may be placed.

According to the embodiments of the present application, when the trypsin detection kit is used for trypsin detection, the method comprises the following steps:

after the trypsin detection film is prepared, the color of the trypsin detection film before detection can be recorded by means of photographing, or the trypsin detection film can be compared with standard colors on the trypsin detection kit for recording;

then, the trypsin detection film is immersed in a trypsin solution, and after several seconds, it can be observed that the color of the trypsin detection film changes slowly, for example, the color of the trypsin detection film changes from blue to green. After the color is stable, the trypsin detection film is taken out, the color of the trypsin detection film after detection is recorded, and the color of the trypsin detection film after detection is compared with the color of the trypsin detection film before detection for analysis and judgment, or the color of the trypsin detection film can be compared with a color card number for verification, or can be compared with a standard color scale or a standard colorimetric card in the trypsin detection kit, and the change of color depth corresponds to different trypsin concentrations.

Where, the standard color scale or standard colorimetric card for trypsin detection can be prepared by the following method:

preparing trypsin solutions with a range of concentrations, such as 0 ug/mL, 0.5 ug/mL, 5 ug/mL, 10 ug/mL, 25 ug/mL and 100 ug/mL;

respectively taking the above trypsin solutions with various concentrations and placing them into colorimetric vessels, respectively adding the same trypsin detection films, standing for a certain period of time, taking out the trypsin detection films after the color is stable, and recording color results of the trypsin detection films after detection with image capturing equipment such as a camera. After capturing the images, the images with color gradient are used to form a standard color scale for trypsin detection or (through computer processing) are printed to make a standard colorimetric card for trypsin detection.

In addition, if it is not sensitive to color, a color analysis software can be used to analyze the color of the trypsin detection film. Specifically, the color of the trypsin detection film can be quantified by using color space knowledge, for example, HSI value or RGB value can be obtained by using MATLAB software (the value fluctuates to a certain extent due to the influence of environmental factors). The RGB color values contrast relationship of the trypsin detection film is shown in FIG. 1 .

It can be seen from the data in FIG. 1 that the RGB values change obviously, which can clearly reflect the color change of the trypsin detection film after staining of trypsin with different concentrations, so that the concentration of trypsin can be determined.

It can be understood that the RGB model is a commonly used color information expression mode at present, and it uses the brightness of the three primary colors of red, green and blue to quantitatively express color. This model is also called additive color mixing model, that is, a method to realize color mixing by superimposing RGB colors, and that any color in RGB color space can be represented by a point in three-dimensional space. Therefore, the color change can be quantized by RGB value and determined. After collecting the images of the trypsin detection film, the RGB color analyzer can be used or the images can be imported into the color analysis software, such as MATLAB, to quantify the color values for comparison, in which case the standard colorimetric card is not required. Alternatively, a standard colorimetric card can be prepared in advance for comparison, and only comparison with the standard colorimetric card is required without import analysis.

In order to facilitate the understanding of the present application, the present application can be further described in combination with specific examples below. In the following embodiments, the raw materials used are commercially available unless otherwise specified.

Embodiment 1

The preparation of the trypsin detection film comprises the following steps:

taking bromobutane and vinylimidazole in equal molar ratio, placing in a glass bottle, and performing ultrasonic concussion for 15 min until the two are fully mixed. Then, after removing impurities, adding acrylonitrile solution with the same mass as bromobutane and vinylimidazole, then adding MBA with a mass ratio of 10% of the total mass of bromobutane, vinylimidazole and acrylonitrile and TPO with a mass ratio of 2% of the total mass of bromobutane, vinylimidazole and acrylonitrile, and then performing ultrasonic concussion for 30 min to form clear and transparent film-forming solution.

Preparing a glass plate, wetting the glass plate with water, sticking a tinfoil on the surface of the glass plate, with the smooth surface of the tinfoil facing upward, wiping it with a lint-free cloth until there is no wrinkle. Applying the first lubricant, white vaseline, to the tinfoil, and wiping with the lint-free cloth until the surface is smooth. Then, putting the film-forming solution (may using a pipette) on the tinfoil with white vaseline (the thickness of the final synthesized film can be adjusted by adjusting the volume of the added film-forming solution), and then slowly pressing the glass cover plate with the contact surface uniformly coated with the second lubricant, white vaseline.

Then, irradiating with 250 nm ultraviolet light for 15 min in a polymerization reaction chamber to make the film-forming solution undergo polymerization reaction to form a transparent film. After 5 minutes in a curing chamber, removing and placing at room temperature for 15 minutes. Then, removing the base plate assembly, placing the glass cover plate with transparent film in water (adding water to cover the transparent film), and placing it for 20 min, so that the transparent film is automatically separated from the glass cover plate, and the complete transparent film floats on the water surface. Performing ultrasonic cleaning of the obtained transparent film in clear water, absolute ethyl alcohol and clear water in sequence to obtain the polymer film substrate.

Immersing the polymer film substrate in 1 mg/mL of bromocresol purple dye solution (the solvent of the dye solution is absolute ethyl alcohol), placing it at 30° C. for 20 min, and then taking it out. At this point, the film is blue. Then performing ultrasonic cleaning with clean water-absolute ethyl alcohol-clean water to obtain the trypsin detection film with bromocresol purple dye, and storing it at room temperature.

Embodiment 2

The preparation of the trypsin detection film is different from that of embodiment 1 only in that:

Immersing the polymer film substrate in 2 mg/mL of 3, 3′, 5, 5′-tetramethylbenzidine dye solution (the solvent of the dye solution is ethyl alcohol solution with mass concentration of 80%), placing it at 35° C. for 15 min, and then taking out the film. At this point, the film is colorless. Then performing ultrasonic cleaning with clear water-absolute ethyl alcohol-clear water, and storing it at room temperature.

Embodiment 3

The preparation of the trypsin detection film is different from that of embodiment 1 only in that:

Immersing the polymer film substrate in 2.5 mg/mL mixed dye solution of bromocresol purple dye and xylenol orange dye (the ratio of the two dyes can be 1:1, 1:2, 1:3 or 1:4) (the solvent of the dye solution is ethyl alcohol solution with mass concentration of 80%), placing it at 28° C. for 22 min, and then taking out the film. At this point, the film is purple black. Then performing ultrasonic cleaning with clear water-absolute ethyl alcohol-clear water, and then drying it in an oven at 40° C.T for 15 min for later use.

Embodiment 4

The preparation of the trypsin detection film comprises the following steps:

taking bromobutane and vinylimidazole with a molar ratio of 2:1, placing in a glass bottle, and performing ultrasonic concussion for 20 min until the two are fully mixed. After removing impurities, adding acrylonitrile solution with the same mass as bromobutane and vinylimidazole, then adding MBA with a mass ratio of 8% of the total mass of bromobutane, vinylimidazole and acrylonitrile and TPO with a mass ratio of 1% of the total mass of bromobutane, vinylimidazole and acrylonitrile, and then performing ultrasonic concussion for 30 min to form clear and transparent film-forming solution.

Preparing a glass plate, wetting the glass plate with water, sticking a tinfoil on the surface of the glass plate, with the smooth surface of the tinfoil facing upward, wiping it with a lint-free cloth until there is no wrinkle. Applying the first lubricant, white vaseline, to the tinfoil, and continuing to wipe with the lint-free cloth until the surface is smooth. Then, putting the film-forming solution (may using a pipette) on the tinfoil with white vaseline (the thickness of the final synthesized film can be adjusted by adjusting the volume of the added film-forming solution, for example, 20-50 μm), and then slowly pressing the glass cover plate with the contact surface uniformly coated with the second lubricant, white vaseline.

Then, irradiating with 300 nm ultraviolet light for 15 min in the polymerization reaction chamber to make the film-forming solution undergo polymerization reaction to form a transparent film. Then, placing in a curing chamber for 10 minutes, and removing and placing at room temperature for 15 minutes. Then, removing the base plate assembly, placing the glass cover plate with transparent film in water (adding water to cover the transparent film), and placing it for 25 min, so that the transparent film is automatically separated from the glass cover plate, and the complete transparent film floats on the water surface.

Performing ultrasonic cleaning of the obtained transparent film in clear water, absolute ethyl alcohol and clear water in sequence to obtain the polymer film substrate.

Immersing the polymer film substrate in 1 mg/mL bromocresol purple dye solution (the solvent of the dye solution is absolute ethyl alcohol), placing it at 30° C. for 20 min, then take out the film. At this point, the film is blue. Then performing ultrasonic cleaning with clean water-absolute ethyl alcohol-clean water, and storing it at room temperature.

Embodiment 5

The preparation of the trypsin detection film comprises the following steps:

taking bromobutane and vinylimidazole with a molar ratio of 1.2:1, placing in a glass bottle, and performing ultrasonic concussion for 25 min until the two are fully mixed. After removing impurities, adding acrylonitrile solution with the same mass as bromobutane and vinylimidazole, then adding MBA with a mass ratio of 12% of the total mass of bromobutane, vinylimidazole and acrylonitrile and TPO with a mass ratio of 4% of the total mass of bromobutane, vinylimidazole and acrylonitrile, and then performing ultrasonic concussion for 20 min to form clear and transparent film-forming solution.

Preparing a glass plate, wetting the glass plate with water, sticking a tinfoil on the surface of the glass plate, with the smooth surface of the tinfoil facing upward, wiping it with a lint-free cloth until there is no wrinkle. Applying the first lubricant, silicone oil, to the tinfoil, and continuing to wipe with the lint-free cloth until the surface is smooth. Then, putting the film-forming solution (may using a pipette) on the tinfoil with silicone oil, and then slowly pressing the glass cover plate with the contact surface uniformly coated with the second lubricant, white vaseline.

Then, irradiating with 265 nm ultraviolet light for 18 min in a polymerization reaction chamber to make the film-forming solution undergo polymerization reaction to form a transparent film; then, placing in a curing chamber for 7 minutes and then removing and placing at room temperature for 15 minutes. Then, removing the base plate assembly, placing the glass cover plate with transparent film in water (adding water to cover the transparent film), and placing it for 20 min, so that the transparent film is automatically separated from the glass cover plate, and the complete transparent film floats on the water surface. Performing ultrasonic cleaning of the obtained transparent film in clear water, absolute ethyl alcohol and clear water in sequence to obtain the polymer film substrate.

Immersing the polymer film substrate in 2 mg/mL of 3, 3′, 5, 5′-tetramethylbenzidine dye solution (the solvent of dye solution is ethyl alcohol solution with mass concentration of 80%), placing it at 30° C. for 20 min, and then taking out the film. At this point, the film is colorless. Then performing ultrasonic cleaning with clear water-absolute ethyl alcohol-clear water, and storing it at room temperature.

Embodiment 6

The preparation of the trypsin detection film comprises the following steps:

taking bromobutane and vinylimidazole with a molar ratio of 1:1, placing in a glass bottle, and performing ultrasonic concussion for 15 min until the two are fully mixed. After removing impurities, adding acrylonitrile solution with 1.2 times of the total mass of bromobutane and vinylimidazole, then adding MBA with a mass ratio of 10% of the total mass of bromobutane, vinylimidazole and acrylonitrile and TPO with a mass ratio of 3% of the total mass of bromobutane, vinylimidazole and acrylonitrile, and then performing ultrasonic concussion for 30 min to form clear and transparent film-forming solution.

Preparing a smooth stainless steel plate, wetting the stainless steel plate with water, sticking the tinfoil on the surface of the stainless steel plate, and with the smooth surface of the tinfoil facing upward, wiping with a lint-free cloth until there is no wrinkle. Applying the first lubricant, white vaseline, to the tinfoil, and continuing to wipe with the lint-free cloth until the surface is smooth. Then, putting the film-forming solution (may using a pipette) on the tinfoil with white vaseline, and then slowly pressing the cover plate with the contact surface uniformly coated with the second lubricant, grease.

Then, irradiating with 400 nm ultraviolet light for 15 min in a polymerization reaction chamber to make the film-forming solution undergo polymerization reaction to form a transparent film; then, placing in a curing chamber for 5 minutes and then removing and placing at room temperature for 15 minutes. Then, removing the glass cover plate and base plate, putting the tinfoil with transparent film into water (adding water to submerge the transparent film), and placing for 30 min, so that the transparent film is automatically separated from the tinfoil, and the complete transparent film floats on the water surface; performing ultrasonic cleaning of the obtained transparent film in clear water, absolute ethyl alcohol and clear water in sequence, so as to obtain the polymer film substrate.

Immersing the polymer film substrate in 0.5 mg/mL bromocresol purple dye solution (the solvent of the dye solution is absolute ethyl alcohol), placing it at 30° C. for 20 min, then taking out the film. At this point, the film is light blue. Then performing ultrasonic cleaning with clean water-absolute ethyl alcohol-clean water, and storing it at room temperature.

Embodiment 7

The preparation of the trypsin detection film comprises the following steps:

taking bromobutane and vinylimidazole in equal molar ratio, placing in a glass bottle, and performing ultrasonic concussion for 15 min until the two are well mixed. Then, after removing impurities, adding acrylonitrile solution with the same mass as bromobutane and vinylimidazole, then adding MBA with a mass ratio of 10% of the total mass of bromobutane, vinylimidazole and acrylonitrile and TPO with a mass ratio of 2% of the total mass of bromobutane, vinylimidazole and acrylonitrile, and then performing ultrasonic concussion for 30 min to form clear and transparent film-forming solution.

Preparing a glass plate as the base plate, applying the first lubricant, white vaseline, on the glass plate, and continuing to wipe it with a lint-free cloth until the surface is smooth. Then, putting the film-forming solution (may using a pipette) on the base plate with white vaseline (the thickness of the final synthesized film can be adjusted by adjusting the volume of the added film-forming solution), and then slowly pressing the glass cover plate with the contact surface uniformly coated with the second lubricant, white vaseline.

The rest is the same as the embodiment 1. The main difference between this embodiment and embodiment 1 is that the base plate assembly does not include the tinfoil in this embodiment.

Application Embodiment 1

The trypsin detection film prepared in the above embodiments is applied to the actual trypsin detection. Taking embodiment 1 as an example, when the trypsin detection film obtained in embodiment 1 is applied to the trypsin detection, the specific detection method comprises:

cutting the obtained detection film into a size of lens for traditional enteroscopy and an annular structure, where the specific size can be determined according to the scattering angle of the lens and the lens barrel, and generally, the optical inspection of the enteroscopy is not affected as much as possible, and the detection film can be observed at the same time;

fixing the detection film onto the lens, which goes inside the human body along with a intubation tube for detection;

after reaching a corresponding area, firstly observe whether the detection film changes; if the corresponding area has diseases, the internal environment and the color of the film change, comparing the change value with the standard colorimetric card for trypsin detection, so as to determine the concentration of trypsin at this time.

The person skilled in the art realizes that the present application by no means is limited to the preferred embodiments described above. On the contrary, many modifications and variations are possible within the scope of the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this disclosure are intended to be included within the scope of the present application.

It should be noted that part of the patent application document contains the content protected by copyright. In addition to making copies of the patent documents of the Patent Office or recorded patent documents, the copyright is reserved by the copyright owner. 

1. A preparation method for a trypsin detection film, which is for trypsin detection, the method comprising the following steps: providing a polymer film substrate; immersing the polymer film substrate in a dye solution to attach a dye to the polymer film substrate, so as to obtain the trypsin detection film; wherein the dye interacts with trypsin, and the color of the dye is different in an environment with different trypsin concentration; the dye comprises at least one of a bromocresol purple dye, a 3, 3′, 5, 5′-tetramethylbenzidine dye, a triarylmethane dye, a xylenol orange dye or a metal complex dye; providing a base plate assembly comprising a base plate and a first lubricant disposed on the base plate; placing a film-forming solution of the polymer film substrate on the base plate assembly, and putting a cover plate with a second lubricant on the film-forming solution, wherein the film-forming solution is in contact with the first lubricant and the second lubricant, respectively; and separating the polymer film substrate from the base plate assembly and the cover plate after a polymerization reaction of various raw materials in the film-forming solution to obtain the polymer film substrate.
 2. The preparation method of claim 1, wherein the concentration of the dye solution is 0.25-2.5 mg/mL or 1-2 mg/mL; or, a solvent of the dye solution comprises water and/or an alcoholic solvent.
 3. The preparation method of claim 1, wherein the temperature for immersing the polymer film substrate in the dye solution is 20-40° C. or 25-35° C., or the time for immersing the polymer film substrate in the dye immersing is 15-30 min or 18-22 min.
 4. (canceled)
 5. The preparation method of claim 4, wherein the base plate assembly further comprises a tinfoil, wherein the tinfoil is arranged on the base plate, and the first lubricant is arranged on the tinfoil.
 6. The preparation method of claim 4, wherein the first lubricant and the second lubricant are each independently selected from at least one of white vaseline, silicone oil, paraffin, mineral oil or grease.
 7. The preparation method of claim 4, wherein the polymerization reaction is carried out under the irradiation of ultraviolet light, wherein the wavelength of the ultraviolet light is 250-400 nm, and/or the irradiation time of the ultraviolet light is 10-30 min; or, wherein the polymer film substrate is placed in a curing chamber for 5-10 min after ultraviolet irradiation.
 8. The preparation method of claim 4, wherein the method for separating the polymer film substrate from the base plate assembly and the cover plate comprises: removing the base plate assembly, placing the cover plate attached with the polymer film substrate in a standing solution, standing for 10-30 min, and removing the cover plate to obtain the polymer film substrate; or, removing the cover plate, placing the base plate assembly attached with the polymer film substrate in the standing solution, standing for 10-30 min, and removing the base plate assembly to obtain the polymer film substrate; or, removing the base plate, placing the cover plate and a tinfoil attached with the polymer film substrate in the standing solution, standing for 10-30 min, and removing the tinfoil to obtain the polymer film substrate.
 9. The preparation method of claim 8, wherein after separating the polymer film substrate from the base plate assembly and the cover plate, the method further comprises the step of cleaning the polymer film substrate, comprising ultrasonic cleaning in clear water, an alcohol solution and clear water in sequence.
 10. The preparation method of claim 1, wherein the preparation of the film-forming solution comprises: uniformly mixing an ionic liquid monomer and a base film monomer, adding a cross-linking agent and an initiator, and then carrying out a second ultrasonic treatment to obtain the film-forming solution; and the irradiation time of the second ultrasonic treatment is 10-30 min.
 11. The preparation method of claim 10, wherein the preparation of the film-forming solution further comprises performing a first ultrasonic treatment on the ionic liquid monomer, wherein the first ultrasonic treatment lasts for 10-30 min.
 12. The preparation method of claim 10, wherein the film-forming solution comprises at least one of an imidazole ionic liquid, a pyridine ionic liquid, a quaternary ammonium salt ionic liquid, a quaternary phosphine ionic liquid or a pyrrolidine ionic liquid; or, the ionic liquid monomer comprises bromobutane and vinylimidazole, or a molar ratio of bromobutane to vinylimidazole is 2:1 to 1:1; or, the base film monomer comprises acrylonitrile, or the mass of the acrylonitrile is greater than or equal to the sum of the mass of the bromobutane and the vinylimidazole; or, the cross-linking agent comprises N, N′-methanediylbisprop-2-enamide, or the mass of the cross-linking agent is 8 wt %-12 wt % of the total mass of the bromobutane, the vinylimidazole and the acrylonitrile; or, the initiator comprises Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, or the mass of the initiator is 1 wt %-4 wt % of the total mass of the bromobutane, the vinylimidazole and the acrylonitrile.
 13. A trypsin detection film, which is prepared by the preparation method for the trypsin detection film according to claim
 1. 14. An application of a detection film prepared by the preparation method for the trypsin detection film according to claim 1 in detection of trypsin, wherein the application comprises: placing the trypsin detection film in a liquid to be detected, so that the dye in the trypsin detection film contacts or interacts with the liquid to be detected.
 15. The application of claim 14, wherein the application further comprises: observing a color change of the trypsin detection film.
 16. The application of claim 15, wherein the application further comprises: determining the concentration of the trypsin according to different colors shown by the trypsin detection film, wherein the trypsin detection film has different colors in an environment of the liquid to be detected containing different concentration of trypsin. 17-18. (canceled) 